Mechanical overload decreases tenogenic differentiation compared to physiological load in bioartificial tendons.

BACKGROUND: Tenocytes as specialised fibroblasts and inherent cells of tendons require mechanical load for their homeostasis. However, how mechanical overload compared to physiological load impacts on the tenogenic differentiation potential of fibroblasts is largely unknown. METHODS: Three-dimensional bioartificial tendons (BATs) seeded with murine fibroblasts (cell line C3H10T1/2) were subjected to uniaxial sinusoidal elongation at either overload conditions (0-16%, Ø 8%) or physiological load (0-8%, Ø 4%). This regime was applied for 2 h a day at 0.1 Hz for 7 days. Controls were unloaded, but under static tension. RESULTS: Cell survival did not differ among overload, physiological load and control BATs. However, gene expression of tenogenic and extra-cellular matrix markers (Scx, Mkx, Tnmd, Col1a1 and Col3a1) was significantly decreased in overload versus physiological load and controls, respectively. In contrast, Mmp3 was significantly increased at overload compared to physiological load, and significantly decreased under physiological load compared to controls. Mkx and Tnmd were significantly increased in BATs subjected to physiological load compared to controls. Proinflammatory interleukin-6 showed increased protein levels comparing load (both over and physiological) versus unloaded controls. Alignment of the cytoskeleton in strain direction was decreased in overload compared to physiological load, while other parameters such as nuclear area, roundness or cell density were less affected. CONCLUSIONS: Mechanical overload decreases tenogenic differentiation and increases ECM remodelling/inflammation in 3D-stimulated fibroblasts, whereas physiological load may induce opposite effects.

Sites of chromosomal instability in the context of nuclear architecture and function.

Chromosomal fragile sites are described as areas within the tightly packed mitotic chromatin that appear as breaks or gaps mostly tracing back to a loosened structure and not a real nicked break within the DNA molecule. Most facts about fragile sites result from studies in mitotic cells, mainly during metaphase and mainly in lymphocytes. Here, we synthesize facts about the genomic regions that are prone to form gaps and breaks on metaphase chromosomes in the context of interphase. We conclude that nuclear architecture shapes the activity profile of the cell, i.e. replication timing and transcriptional activity, thereby influencing genomic integrity during interphase with the potential to cause fragility in mitosis. We further propose fragile sites as examples of regions specifically positioned in the interphase nucleus with putative anchoring points at the nuclear lamina to enable a tightly regulated replication-transcription profile and diverse signalling functions in the cell. Consequently, fragility starts before the actual display as chromosomal breakage in metaphase to balance the initial contradiction of cellular overgrowth or malfunctioning and maintaining diversity in molecular evolution.

The fate of chlorophyll in phytophagous insects goes beyond nutrition.

Chlorophyll (Chl) is a natural compound that is found in all autotrophic plants. Since phytophagous insects ingest the photosynthetically active material with the plant leaves, the question arises if and how herbivores deal with Chl and its degradation products. Here we review findings on Chl degradation in phytophagous insects and highlight the role of these ubiquitous plant metabolites for plant-feeding insects. Due to the anaerobic gut of many insects, the degradation is limited to the removal of the peripheral substituents, while the tetrapyrrole core remains intact. Proteins, such as red fluorescent protein, P252 (a novel 252-kDa protein), and chlorophyllide binding protein have been reported to occur in the insect gut and might be indirectly connected to Chl degradation. Besides of an nutritional value, e.g., by taking up Mg2+ ions or by sequestration of carbon from the phytol side chain, the Chl degradation products may serve the insect, after binding to certain proteins, as antimicrobial, antifungal, and antiviral factors. The protein complexes may also confer protection against reactive oxygen species. The antibiotic potential of proteins and degradation products does not only benefit phytophagous insects but also human being in medical application of cancer treatment for instance. This review highlights these aspects from a molecular, biochemical, and ecological point of view.

Autofluorescence-Based Identification and Functional Validation of Antennal Gustatory Sensilla in a Specialist Leaf Beetle.

Herbivorous insects mainly rely on their sense of taste to decode the chemical composition of potential hosts in close range. Beetles for example contact and scan leaves with their tarsi, mouthparts and antennal tips, i.e., appendages equipped with gustatory sensilla, among other sensillum types. Gustatory neurons residing in such uniporous sensilla detect mainly non-volatile compounds that contribute to the behavioral distinction between edible and toxic plants. However, the identification of gustatory sensilla is challenging, because an appendage often possesses many sensilla of distinct morphological and physiological types. Using the specialized poplar leaf beetle (Chrysomela populi, Chrysomelidae), here we show that cuticular autofluorescence scanning combined with electron microscopy facilitates the identification of antennal gustatory sensilla and their differentiation into two subtypes. The gustatory function of sensilla chaetica was confirmed by single sensillum tip-recordings using sucrose, salicin and salt. Sucrose and salicin were found at higher concentrations in methanolic leaf extracts of poplar (Populus nigra) as host plant compared to willow (Salix viminalis) as control, and were found to stimulate feeding in feeding choice assays. These compounds may thus contribute to the observed preference for poplar over willow leaves. Moreover, these gustatory cues benefited the beetle's performance since weight gain was significantly higher when C. populi were reared on leaves of poplar compared to willow. Overall, our approach facilitates the identification of insect gustatory sensilla by taking advantage of their distinct fluorescent properties. This study also shows that a specialist beetle selects the plant species that provides optimal development, which is partly by sensing some of its characteristic non-volatile metabolites via antennal gustatory sensilla.

A subset of chemosensory genes differs between two populations of a specialized leaf beetle after host plant shift.

Due to its fundamental role in shaping host selection behavior, we have analyzed the chemosensory repertoire of Chrysomela lapponica. This specialized leaf beetle evolved distinct populations which shifted from the ancestral host plant, willow (Salix sp., Salicaceae), to birch (Betula rotundifolia, Betulaceae). We identified 114 chemosensory candidate genes in adult C. lapponica: 41 olfactory receptors (ORs), eight gustatory receptors, 17 ionotropic receptors, four sensory neuron membrane proteins, 32 odorant binding proteins (OBPs), and 12 chemosensory proteins (CSP) by RNA-seq. Differential expression analyses in the antennae revealed significant upregulation of one minus-C OBP (Clap OBP27) and one CSP (Clap CSP12) in the willow feeders. In contrast, one OR (Clap OR17), four minus-C OBPs (Clap OBP02, 07, 13, 20), and one plus-C OBP (Clap OBP32) were significantly upregulated in birch feeders. The differential expression pattern in the legs was more complex. To narrow down putative ligands acting as cues for host discrimination, the relative abundance and diversity of volatiles of the two host plant species were analyzed. In addition to salicylaldehyde (willow-specific), both plant species differed mainly in their emission rate of terpenoids such as (E,E)-α-farnesene (high in willow) or 4,8-dimethylnona-1,3,7-triene (high in birch). Qualitatively, the volatiles were similar between willow and birch leaves constituting an "olfactory bridge" for the beetles. Subsequent structural modeling of the three most differentially expressed OBPs and docking studies using 22 host volatiles indicated that ligands bind with varying affinity. We suggest that the evolution of particularly minus-C OBPs and ORs in C. lapponica facilitated its host plant shift via chemosensation of the phytochemicals from birch as novel host plant.

Silencing cuticular pigmentation genes enables RNA FISH in intact insect appendages.

Optical imaging of gene expression by fluorescence in situ hybridisation (FISH) in insects is often impeded by their pigmented cuticle. As most chemical bleaching agents are incompatible with FISH, we developed an RNA interference (RNAi)-based method for clearing cuticular pigmentation which enables the use of whole-mount body appendages for RNA FISH (termed RNA-i-FISH). Silencing laccase2 or tyrosine hydroxylase in two leaf beetles species (Chrysomela populi and Phaedon cochleariae) cleared their pigmented cuticle and decreased light absorbance. Subsequently, intact appendages (palps, antennae, legs) from RNAi-cleared individuals were used to image the expression and spatial distribution of antisense mRNA of two chemosensory genes encoding gustatory receptor and odorant-binding protein. Imaging did not work for RNAi controls because the pigmentation was retained, or for FISH controls (sense mRNA). Several bleaching agents were incompatible with FISH, because of degradation of RNA, lack of clearing efficacy or long incubation times. Overall, silencing pigmentation genes is a significant improvement over bleaching agents, enabling FISH in intact insect appendages.

Spatial separation of the cyanogenic ß-glucosidase ZfBGD2 and cyanogenic glucosides in the haemolymph of Zygaena larvae facilitates cyanide release.

Low molecular weight compounds are typically used by insects and plants for defence against predators. They are often stored as inactive ß-glucosides and kept separate from activating ß-glucosidases. When the two components are mixed, the ß-glucosides are hydrolysed releasing toxic aglucones. Cyanogenic plants contain cyanogenic glucosides and release hydrogen cyanide due to such a well-characterized two-component system. Some arthropods are also cyanogenic, but comparatively little is known about their system. Here, we identify a specific ß-glucosidase (ZfBGD2) involved in cyanogenesis from larvae of Zygaena filipendulae (Lepidoptera, Zygaenidae), and analyse the spatial organization of cyanide release in this specialized insect. High levels of ZfBGD2 mRNA and protein were found in haemocytes by transcriptomic and proteomic profiling. Heterologous expression in insect cells showed that ZfBGD2 hydrolyses linamarin and lotaustralin, the two cyanogenic glucosides present in Z. filipendulae. Linamarin and lotaustralin as well as cyanide release were found exclusively in the haemoplasma. Phylogenetic analyses revealed that ZfBGD2 clusters with other insect ß-glucosidases, and correspondingly, the ability to hydrolyse cyanogenic glucosides catalysed by a specific ß-glucosidase evolved convergently in insects and plants. The spatial separation of the ß-glucosidase ZfBGD2 and its cyanogenic substrates within the haemolymph provides the basis for cyanide release in Z. filipendulae. This spatial separation is similar to the compartmentalization of the two components found in cyanogenic plant species, and illustrates one similarity in cyanide-based defence in these two kingdoms of life.

Contact chemosensation of phytochemicals by insect herbivores.

Contact chemosensation, or tasting, is a complex process governed by nonvolatile phytochemicals that tell host-seeking insects whether they should accept or reject a plant. During this process, insect gustatory receptors (GRs) contribute to deciphering a host plant's metabolic code. GRs recognise many different classes of nonvolatile compounds; some GRs are likely to be narrowly tuned and others, broadly tuned. Although primary and/or secondary plant metabolites influence the insect's feeding choice, their decoding by GRs is challenging, because metabolites in planta occur in complex mixtures that have additive or inhibitory effects; in diverse forms composed of structurally unrelated molecules; and at different concentrations depending on the plant species, its tissue and developmental stage. Future studies of the mechanism of insect herbivore GRs will benefit from functional characterisation taking into account the spatio-temporal dynamics and diversity of the plant's metabolome. Metabolic information, in turn, will help to elucidate the impact of single ligands and complex natural mixtures on the insect's feeding choice.

Lepidopteran defence droplets - a composite physical and chemical weapon against potential predators.

Insects often release noxious substances for their defence. Larvae of Zygaena filipendulae (Lepidoptera) secrete viscous and cyanogenic glucoside-containing droplets, whose effectiveness was associated with their physical and chemical properties. The droplets glued mandibles and legs of potential predators together and immobilised them. Droplets were characterised by a matrix of an aqueous solution of glycine-rich peptides (H-WG11-NH2) with significant amounts of proteins and glucose. Among the proteins, defensive proteins such as protease inhibitors, proteases and oxidases were abundant. The neurotoxin ß-cyanoalanine was also found in the droplets. Despite the presence of cyanogenic glucosides, which release toxic hydrogen cyanide after hydrolysis by a specific ß-glucosidase, the only ß-glucosidase identified in the droplets (ZfBGD1) was inactive against cyanogenic glucosides. Accordingly, droplets did not release hydrogen cyanide, unless they were mixed with specific ß-glucosidases present in the Zygaena haemolymph. Droplets secreted onto the cuticle hardened and formed sharp crystalline-like precipitates that may act as mandible abrasives to chewing predators. Hardening followed water evaporation and formation of antiparallel ß-sheets of the peptide oligomers. Consequently, after mild irritation, Zygaena larvae deter predators by viscous and hardening droplets that contain defence proteins and ß-cyanoalanine. After severe injury, droplets may mix with exuding haemolymph to release hydrogen cyanide.

Metabolism, excretion and avoidance of cyanogenic glucosides in insects with different feeding specialisations.

Cyanogenic glucosides (CNglcs) are widespread plant defence compounds releasing toxic hydrogen cyanide when hydrolysed by specific ß-glucosidases after plant tissue damage. In contrast to specialist herbivores that have mechanisms to avoid toxicity from CNglcs, it is generally assumed that non-adapted herbivores are negatively affected by CNglcs. Recent evidence, however, implies that the defence potential of CNglcs towards herbivores may not be as effective as previously anticipated. Here, performance, metabolism and excretion products of insects not adapted to CNglcs were analysed, including species with different degrees of dietary specialisation (generalists, specialists) and different feeding modes (leaf-snipping lepidopterans, piercing-sucking aphids). Insects were reared either on cyanogenic or acyanogenic plants or on an artificial cyanogenic diet. Lepidopteran generalists (Spodoptera littoralis, Spodoptera exigua, Mamestra brassicae) were compared to lepidopteran glucosinolate-specialists (Pieris rapae, Pieris brassicae, Plutella xylostella), and a generalist aphid (Myzus persicae) was compared to an aphid glucosinolate-specialist (Lipaphis erysimi). All insects were tolerant to cyanogenic plants; in lepidopterans tolerance was mainly due to excretion of intact CNglcs. The two Pieris species furthermore metabolized aromatic CNglcs to amino acid conjugates (Cys, Gly, Ser) and derivatives of these, which is similar to the metabolism of benzylglucosinolates in these species. Aphid species avoided uptake of CNglcs during feeding. Our results imply that non-adapted insects tolerate plant CNglcs either by keeping them intact for excretion, metabolizing them, or avoiding uptake.

How insects overcome two-component plant chemical defence: plant ß-glucosidases as the main target for herbivore adaptation.

Insect herbivory is often restricted by glucosylated plant chemical defence compounds that are activated by plant ß-glucosidases to release toxic aglucones upon plant tissue damage. Such two-component plant defences are widespread in the plant kingdom and examples of these classes of compounds are alkaloid, benzoxazinoid, cyanogenic and iridoid glucosides as well as glucosinolates and salicinoids. Conversely, many insects have evolved a diversity of counteradaptations to overcome this type of constitutive chemical defence. Here we discuss that such counter-adaptations occur at different time points, before and during feeding as well as during digestion, and at several levels such as the insects' feeding behaviour, physiology and metabolism. Insect adaptations frequently circumvent or counteract the activity of the plant ß-glucosidases, bioactivating enzymes that are a key element in the plant's two-component chemical defence. These adaptations include host plant choice, non-disruptive feeding guilds and various physiological adaptations as well as metabolic enzymatic strategies of the insect's digestive system. Furthermore, insect adaptations often act in combination, may exist in both generalists and specialists, and can act on different classes of defence compounds. We discuss how generalist and specialist insects appear to differ in their ability to use these different types of adaptations: in generalists, adaptations are often inducible, whereas in specialists they are often constitutive. Future studies are suggested to investigate in detail how insect adaptations act in combination to overcome plant chemical defences and to allow ecologically relevant conclusions.

The multiple strategies of an insect herbivore to overcome plant cyanogenic glucoside defence.

Cyanogenic glucosides (CNglcs) are widespread plant defence compounds that release toxic hydrogen cyanide by plant ß-glucosidase activity after tissue damage. Specialised insect herbivores have evolved counter strategies and some sequester CNglcs, but the underlying mechanisms to keep CNglcs intact during feeding and digestion are unknown. We show that CNglc-sequestering Zygaena filipendulae larvae combine behavioural, morphological, physiological and biochemical strategies at different time points during feeding and digestion to avoid toxic hydrolysis of the CNglcs present in their Lotus food plant, i.e. cyanogenesis. We found that a high feeding rate limits the time for plant ß-glucosidases to hydrolyse CNglcs. Larvae performed leaf-snipping, a minimal disruptive feeding mode that prevents mixing of plant ß-glucosidases and CNglcs. Saliva extracts did not inhibit plant cyanogenesis. However, a highly alkaline midgut lumen inhibited the activity of ingested plant ß-glucosidases significantly. Moreover, insect ß-glucosidases from the saliva and gut tissue did not hydrolyse the CNglcs present in Lotus. The strategies disclosed may also be used by other insect species to overcome CNglc-based plant defence and to sequester these compounds intact.

Sequestration, tissue distribution and developmental transmission of cyanogenic glucosides in a specialist insect herbivore.

Considering the staggering diversity of bioactive natural products present in plants, insects are only able to sequester a small number of phytochemicals from their food plants. The mechanisms of how only some phytochemicals are sequestered and how the sequestration process takes place remains largely unknown. In this study the model system of Zygaena filipendulae (Lepidoptera) and their food plant Lotus corniculatus is used to advance the knowledge of insect sequestration. Z. filipendulae larvae are dependent on sequestration of the cyanogenic glucosides linamarin and lotaustralin from their food plant, and have a much lower fitness if reared on plants without these compounds. This study investigates the fate of the cyanogenic glucosides during ingestion, sequestration in the larvae, and in the course of insect ontogeny. To this purpose, double-labeled linamarin and lotaustralin were chemically synthesized carrying two stable isotopes, a (2)H labeled aglucone and a (13)C labeled glucose moiety. In addition, a small amount of (14)C was incorporated into the glucose residue. The isotope-labeled compounds were applied onto cyanogenic L. corniculatus leaves that were subsequently presented to the Z. filipendulae larvae. Following ingestion by the larvae, the destiny of the isotope labeled cyanogenic glucosides was monitored in different tissues of larvae and adults at selected time points, using radio-TLC and LC-MS analyses. It was shown that sequestered compounds are taken up intact, contrary to earlier hypotheses where it was suggested that the compounds would have to be hydrolyzed before transport across the gut. The uptake from the larval gut was highly stereo selective as the ß-glucosides were retained while the α-glucosides were excreted and recovered in the frass. Sequestered compounds were rapidly distributed into all analyzed tissues of the larval body, partly retained throughout metamorphosis and transferred into the adult insect where they were distributed to all tissues. During subsequent mating, isotope labeled cyanogenic glucosides were transferred from the male to the female in the nuptial gift.